1. Dr. Edward P. Fox February 26, 2016

2. Chronic Lymphoproliferative disorder Progressive accumulation functionally incompetent lymphocytes Monoclonal Overview

3. Epidemiology Most common leukemia – Western countries 30% in US 16,000 total 9500 male / 6500 females Male:female ratio = 1.7:1 6.75 / 3.65 per 100,000 – US 5.87 / 4.01 per 100,000 - Europe

4. Epidemiology Disease of Elderly Median age of diagnosis – 70 Can be diagnosed in 30-39 year-olds Incidence increases rapidly with age

5. Epidemiology Race Higher in Caucasians Lower in –African Americans - Asian and Pacific Islanders 90% lower in China and Japan

6. Epidemiology Geography Point to genetic rather than environmental factors Japanese in Hawaii – incidence = Japan Japan – cytogenetics and molecular genetics the same

7. Epidemiology Environmental Risks None known No increase in atomic bomb survivors Possibly Rubber manufacturers Farmers Benzene / solvents Multiple pneumonias

8. Epidemiology Family Studies Family members of CLL patients have greater risks: Lymphoid malignancies Hematologic malignancies Solid malignancies 17% of first degree relatives have MBL

9. Epidemiology Multi-Hit Hypothesis Median age parent (72), child (51) DNA analysis of monozygotic twins: Genetically distinct cells Transforming event occurs later in life

10. Clinical Presentation Wide variety of presentations Commonly asymptomatic lymphadenopathy 25% abnormal CBC w/o symptoms

11. Clinical Presentation 10% have “B” symptoms Fevers (>38 o, > 2 weeks, no infection) Sweats (drenching, no infection) Weight loss (≥ 10% in 6 months) Extreme fatigue (ECOG ≥ 2)

12. Clinical Presentation ITP AIHA PRCA Exaggerated reaction to insect bites

13. Clinical Presentation Signs: Lymphadenopathy = 50-90% Splenomegaly = 25-55% Hepatomegaly = 10-25% Cutaneous = <5% Renal = MPGN Rarely GI or CNS

14. Clinical Presentation Laboratory abnormalities Lymphocytosis > 5,000/microL – requisite > 100,000 – 30% > 200,000 – rare, usually normal viscosity

15. Clinical Presentation Labs: Cytopenias – usually mild ITP = 2-3% AIHA = 11% PRCA = 0.5% Agranulocytosis = 0.5%

16. Clinical Presentation Labs: Hypogammaglobulinemia = 8% at diagnosis Usually all 3 classes IgG, A, M Monoclonal paraprotein = 5% Polyclonal increase up to 15%

17. Pathology Initial evaluation should include: CBC Peripheral smear exam Peripheral blood flow cytometry

18. Pathology Not necessary* Bone marrow biopsy / aspirate Lymph node biopsy * but occasionally useful

19. Morphology Lymphocytosis Small mature appearing Dense nucleus No nucleoli, aggregated chromatin Narrow rim of cytoplasm

20. Morphology “Smudge cells” – common Prolymphocytes – small number Larger cells More open, notched nuclei Lacy chromatin, nucleoli

21. Immunophenotyping Key component to diagnosing CLL Cells appear mature Immature functionally and developmentally

22. Immunophenotyping B cell antigens: CD19, CD20, CD23 – present CD21, CD24 – sometimes Co-expression of CD5 – T cell antigen

23. Immunophenotyping Sn Ig : weakly expressed IgM or IgM and IgD Single Ig light chain expressed “light chain restriction” – monoclonal Negative cyclin D1, CD10

24. Immunophenotyping (+)(-) IgMCD10 IgD, IgMCyclin D1 CD19 CD20 CD21 CD5

25. Evaluation and Diagnosis 2008 NCI guidelines: 1. Absolute B lymphocytes >5,000/microL Morphologically mature appearing 2. Flow cytometry demonstrates monoclonality CD19, CD20, CD23 CD5 Kappa or lambda light chain restriction

26. Monoclonal B cell lymphocytosis (MBCL) + flow cytometry Lymphocytosis < 5,000 No adenopathy, hepatomegaly, splenomegaly

27. Small Lymphocytic Lymphoma (SLL) Lymphadenopathy ALC <5,000 No hepatosplenomegaly

28. CLL vs SLL vs MBCL Sometimes fuzzy Controversy ongoing Usually irrelevant to treating patients

29. B-CELL LINEAGE AND DIFFERENTIATION Gaidano et al. Leukemia. 2000;14:563-566. LPL MCL HHV-8 SLL/B-CLL PEL Germinal Center p53 C-MYC BCL-2 FL p53, p16 DLCL Plasma Cells Follicular Mantle Memory B Cells Memory B Cells CD5+ B Cells CD5+ B Cells Marginal Zone BL BCL-6 ? Virgin B Cells Virgin B Cells PAX-5 ? BCL-1 ?

30. Differential Diagnosis Sustained lymphocytosis, monoclonal Mononucleosis Whooping cough - pertussis Toxoplasmosis

31. Differential Diagnosis Monoclonal B cell lymphocytosis (MBCL) Clonal B lymphocytes <5,000 No B symptoms No organomegaly, lymphadenopathy

32. MBCL phenotype 5% of normal CBC’s > 60 y.o. 14% > 60 with lymphocytosis 1-2% per year progression to CLL Virtually 100% CLL patients

33. Prolymphocytic leukemia Larger cells - >90% CD5 negative SmIg normal

34. COMPARISON OF CLL AND PLL CLLPLL sIgfaintstrong CD19+++ CD20+++ CD5++-

35. Mantle Cell Lymphoma May have leukemic phase Cyclin D1, SmIg positive + (11, 14) CD23 negative

36. Others Lymphoplasmacytic lymphoma Hairy cell leukemia Follicular lymphoma Splenic marginal zone lymphoma

37. Genetic Features Apoptosis Used to be felt to be the major cause BCL2 overexpression Cell birth rates > 0.35%/day More likely progress, need treatment

38. Cytogenetics 5 categories of karyotypes (median survival) 17 p deletions (32 months) 11 q deletions (79 months) 12q trisomy (114 months) Normal (111 months) 13 q deletions (133 months)

39. Cytogenetics Abnormalities occur over time Karyotopic evolution – 38% over 5 years Associated with more aggressive disease

40. Cytogenetics 17 p deletions = 10-14% P53 present on 17 p Normally helps trigger apoptosis Poor prognosis More aggressive Less responsive

41. Micro RNA (miRNA) Mutations are common miR-21, miR-155 + nearly 100% CLL Upregulate BCL-2 P53 might regulate miRNA expression

42. Staging Systems RAI vs. BINET RAI l O – lymphocytosis l I – lymphadenopathy l II – hepatosplenomegaly l III – anemia l IV - thrombocytopenia BINET l A - lymphocytosis, < 3 LN groups, nl Hgb and platelets l B - > 3 LN groups l C - anemia, thrombocytopenia

43. Prognosis by Risk Groups RISKRAIBINETSURVIVAL LOWO, I A SAME MED. II B 84 mos HIGHIII, IV C 24 mos

44. Prognosis RAI Risk Group Median Survival 0 L 150 mos Low I L 101 mos Low II I 71 mos Int III H 19mos High IV H 19mos High

45. Prognosis Important distinction in the source of cytopenias Secondary to immune dysfunction = 7.2 years Secondary to marrow infiltration/failure = 2.4 years

46. Prognosis Genetic Abnormalities Risk % Abn High 27 IP53 BIRC3 Int 39 Notch 1 SF3B1 Low +12 or nl Very Low del 13q14 O.S. 5 years10 years 51 29 66 37 78 57 87 69* * same as age matched population

47. Prognosis Others Vitamin D Low Poorer Il-8 High Independent poor CD-20 High Independent poor Free Light Chains Abnormal Poor TPO High Independent MicRNA Low Poor

48. Prognostic Factors - Other Lymphocyte Doubling time # months to double the lymphocyte count < 12 months – aggressive > 12 months - indolent

49. B 2 microglobulin Regulated by exogenous cytokines - ?IL-6 Renal clearance Increased level – poor prognosis

50. IgVH Mutated – better – 50% Unmutated – worse – 50% May relate to developmental stage of B cell Not useful, difficult technically

51. CD38 Present in serum Correlates with IgVH mutation status Higher – poor prognosis

52. IG V GENE MUTATIONS AND CD38: NEWER PROGNOSTIC FACTORS IN CLL Damle et al. Blood. 1999;94:1840-1847. Ig V CD38 Years From Diagnosis Percent Surviving Unmutated Mutated  30% <30% 100 90 80 70 60 50 40 30 20 10 0 20181614121086420 20181614121086420 100 90 80 70 60 50 40 30 20 10 0

53. SURVIVAL CLL BY CD38

54. ZAP 70 Tyrosine kinase Not normally expressed in B cells Strong association with unmutated IgVH

55. Others Vitamin D levels VEGFR-2 receptors TNF Il-8 CD20 TPO miRNA

56. Treatment of CLL Decision to treat Evaluate prognostic factors Period of observation Symptoms

57. Treatment Chemotherapy Fludarabine Fludarabine + Cytoxan Fludarabine + Cytoxan + Rituxan

58. Antibodies 1. Alemtuzumab (Campath) Anti-CD52 Wipes out B cells for awhile Increased risk of infection 2. Rituximab Anti-CD20

59. On RxPost Rx

60. Others Bendamustine Chlorambucil Pentostatin 2-CDA Ibrutinib

61. NEW INITIATIVES IN CLL BCL2 Antisense Proteosome Inhibitor - Velcade Combination Rituxan + Campath Clofarabine (Adenosine Analog) Nelarabine (Guanosine Analog) 2 Methoxy-estradiol: (SOD Inhibitor)

62. Complications of CLL Infections – 50% of CLL deaths Fungi: Candida, Aspergillus, Crypto Bacteria: Listeria, Mycobacteria Viruses: CMV, Zoster, Simplex

63. Infection - Treatment Anti-microbials - ? Prophylaxis IVIG Growth factors – G-CSF

64. Cytopenias Anemia AIHA Marrow suppression/infiltration PRCA GI blood loss

65. Second Cancers Richter’s transformation AML MDS ? Solid tumors

66. Infection – Greater Risk Hypogammaglobulinemia Purine analogs Anti CD52 (Campath), Anti CD20 (Rituxan) Advanced stage diseases

67. Disease related complications B symptoms Symptomatic anemia / thrombocytopenia AIHA, ITP Infectious complications

68. Transformation Richters  DLCL Prolymphocytic Hodgkin’s Multiple Myeloma AML Frequency % 3-7 2 0.5 – 2 0.1 rare

69. THE END Thank you!